Chapter Eighteen:  Chromatography - Paper and Liquid Chromatography

 

 

Chemicals:

 

We dispense #1 and #2 in 500 mL bottles with a 1-hole rubber stopper and a 9 inch plastic pipet

1.   1-propanol/water

      2 parts 1-propanol plus 1 part water

      (eg. 1000 mL 1-propanol plus 500 mL water)

      Note:  You can use 2-propanol instead of 1-propanol.

 

2.   Hexane

 

3.   Food dyes:  1% in water

      a.   Red #3

      b.   Red #40

      c.   Blue #1

      d.   Blue #2

      e.   Yellow #5

      f.    Yellow #6

      g.   Green #3

 

      Food dyes are available in 3 oz. sample sizes from:

 

      Seltzer Chemicals, Inc.

      5931 Priestly Drive

      Carlsbad, CA  92008-3861

      (619-438-0089)

 

      7 dyes, 2 grams each ($15.00 plus $2.00 shipping) are available  from:

 

      Rainbow Colors

      203-871-2033

 

4.   You can make your own shades from the following chart of typical color formulations.  Choose a few.

           

            Shade                                 FD&C Dye                              % of Blend

___________________________________________________________________________

 

            Orange                               Yellow #5                                         95

                                                      Red #40                                              5

 

            Cherry                                Red #40                                            99

                                                      Blue #1                                               1

 

            Strawberry                         Red #40                                            95

                                                      Red #3                                                5

 

            Lemon                                Yellow #5                                       100

 

            Lime                                   Yellow #5                                         95

                                                      Blue #1                                                5

 

            Grape                                 Red #40                                            80

                                                      Blue #1                                             20

 

            Raspberry                          Red #3                                              75

                                                      Yellow #6                                         20

                                                      Blue #1                                                5

 

            Butterscotch                       Yellow #5                                         74

                                                      Red #40                                            24

                                                      Blue #1                                                2

 

            Chocolate                           Red #40                                            52

                                                      Yellow #5                                         40

                                                      Blue #1                                                8

            Caramel                             Yellow #5                                         64

                                                      Red #3                                              21

                                                      Yellow #6                                            9

                                                      Blue #1                                                6

 

            Cinnamon                           Yellow #5                                         60

                                                      Red #40                                            35

                                                      Blue #1                                                5

 

            Peach                                 Red #40                                            60

                                                      Yellow #6                                         40

 

            Cheddar Cheese                 Yellow #5                                         55

                                                      Yellow #6                                         45

 

            Tea, Cola                           Yellow #5                                         70

            or Root Beer                      Red #40                                            25

                                                      Blue #2                                                5

___________________________________________________________________________

 

5.   Commercial food dyes (grocery store)

 

6.   Buffers

      Use a pH meter for accuracy, adding only enough NaOH to reach the desired pH.                               

      Dilute to 100 mL with water.

      A few crystals of thymol will help preserve the solutions.

      a.   pH 7.0

            50 mL 0.1 M KH2PO4

            29 mL 0.1 M NaOH

 

      b.   pH 8.0

            50 mL 0.1 M KH2PO4

            46 mL 0.1 M NaOH

 

      c.   pH 9.0

            50 mL 0.1 M H3BO3/KCl

            21 mL 0.1 M NaOH

 

      d.   pH 10.0

            50 mL 0.1 M H3BO3/KCl

            44 mL 0.1 M NaOH

 

      e.   pH 11.0

            50 mL 0.2 M Na2HPO4

            36 mL 0.1 M NaOH

 

7.   Stock solutions for buffer

      a.   0.1 M KH2PO4:  13.6 grams per     liter

      b.   0.2 M Na2HPO4:  28.4 grams per   liter

 

      c.   0.1 M NaOH:  4 grams per liter

 

 

      d.   0.1 M H3BO3/KCl:  6.2 grams        H3BO3,  7.5 grams KCl per liter

 

8.   Nicotine standard

      1% in water                            HANDLE WITH CARE

      Few drops permanent red ink

 

9.   Dragendorff's reagent (shelf life is 2 weeks or more)

      a.   0.21 grams Bi(NO3)3 in 10 mL        water (not soluble)

 

      b.   4 grams KI in 10 mL water

     

      c.   20 mL glacial acetic acid

 

      Mix a, b, c and add 100 mL water.

 

10.    Silica gel 100-200 mesh (100 grams)

     

11.    0.05 wt.%  bromocresol purple, sodium salt

 

12.    Dye mixture A in water (make fresh each semester)

         0.02% violet #1           

         (Since this has gone to press, Violet #1  is no longer available.  We are attempting to find a substitute.  Until then, methyl red sodium salt can be used)

         0.05% blue #1

         0.05% red #2  (Amaranth)

 

13.    Dye mixture B in water (make fresh each semester)

         0.02% violet #1

         (Since this has gone to press, Violet #1 is no longer available.  We are attempting to find a substitute.  Until then, methyl red sodium salt can be used.)

         0.05% blue #1

         0.05% yellow #5

 

14.    0.1 M acetic acid

         5.7 mL glacial acetic acid per liter

 

15.    30% ethanol

         700 mL water, 300 mL 95% ethanol

 

16.    Beet extract

         Can use fresh beets or juice from canned beets

 

17.    Cigarette extract

         •Weigh 8 grams tobacco and place tobacco into an Erlenmeyer flask

 

         •Add .2 grams Ba(OH)2, 100 mL chloroform, 100 mL water

 

         •Stopper flask and shake vigorously for 3-4 minutes

 

         •Filter the extract using Whatman #41

         •Seperate-save lower layer

         •Evaporate extract to dryness

 

         •Dissolve the residue in 20 mL H2O.  Add enough Ba(OH)2 to make the solution basic (approx. pH 8-9).  The solution may need to be filtered again.

 

 

Supplies:

 

1.   Flair® pens (all colors available)

 

2.   Staplers, eg., Tot® or other small stapler

 

3.   Staples to fit

 

4.   Sheaffer® ink cartridge pens for the nicotine standard and cigarette extract

      •Use a microburet to remove the ink from the cartridge and wash.  (I recommend red ink cartidges, but is not necessary)

 

      •Fill the cartridge with the nicotine standard or cigarette extract

 

      •Make sure the pens are writing

 

      •If you prefer, the cigarette extract can be dispensed in a small container such as one well of a 1 x 12 well microstrip and the students use a plastic applicator to apply the shots to the chromatography paper.

 

      •Whether you use the pen or the plastic applicator, the student needs to touch the tip to the paper long enough to allow the sample to transfer to the paper.  Spotting the cigarette extract twice is probably necessary to get a good chromatograph.

 

      •Take the pens apart when experiment is finished, wash the nibs, empty the cartridges

 

5.   Applicators for spotting the

      chromatograms:   Use the thin, pulled out stem scraps cut from the pulled microburets. (This is the part with the very small diameter that you saved from Chapter 3).  Cut pieces about 1 1/2 inches long.  Each student needs one, but they are easily lost.  Paper applicators are also acceptable.

 

      If you did not save these, or do not have enough, you can make them easily by pulling the stem of the thin- stem pipet to its maximum length.

 

      Plastic toothpicks also work well for spotting the dyes.

 

6.   Chromatography paper, Whatman #1 48 x 57 cm

      Cut pieces 2 " x 7"

      Save the scraps to use in other experiments.  Each sheet will cut 28 pieces, need 8 sheets per 100 students.

 

 

7.   Super jumbo straws

 

8.   Slim straws

 

9.   Punched circles

      Whatman #3 filter paper, 1/4" office punch

 

10.    Permanent marking pen, black, fine point

 

11.    Use 9 inch Beral® plastic pipets:

         a.   Slurry pipets (1 per student):  cut     approximately 4 inches off the   stem

         b.   Plastic scoop (1 per student):  cut    approximately 4 inches off the   stem: cut end of the bulb off at an          angle to make a scoop

 

12.    Polyester fiberfill

         Available at department stores, used to fill pillows

 

13.    To make the pump syringe

         a.   10 cc plastic syringe without            needle

 

         b.   1/8" I. D. Tygon tubing (1/4"           O.D.)

               •Warm in microburner and pull slightly so it will fit tightly inside the super jumbo straw.

               •Keep tension applied until cool

               •Cut in two at the narrow part and then cut each piece about 2-3" long

               •Attach the unpulled end to the syringe where the needle would normally be

               •Check to make sure the pulled end will fit inside the super jumbo straw

Per room:

 

1.   Clock

 

 

Per student:

 

1 -  9 oz. plastic tumbler

 

1 -  Thin-stem pipet

 

1 -  Super jumbo straw

 

1 -  Wooden clothespin

 

1 -  SepCap™ cap, transparent       (with small hole in it)

 

2 -  3 oz. plastic cups

 

1 -  Plastic scoop

 

1 -  Slurry pipet

 

1 -  Pump syringe

 

1 -  Slim straw

 

1 -  Wash bottle

 

 

From Student Kit:

 

1 -  Petri dish

 

1 -  96-well tray, round bottom

 

1 - 24-well tray

 

1 -  1 x12 well microstrip

 

Scissors

 

Office Punch

 

Ruler

 

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